Chk2 is a protein kinase intermediary in DNA damage checkpoint pathways. DNA damage induces phosphorylation of Chk2 at multiple sites concomitant with activation. Chk2 phosphorylated at Thr-68 is found in nuclear foci at sites of DNA damage (1). We report here that Chk2 phosphorylated at Thr-68 and Thr-26 or Ser-28 is localized to centrosomes and midbodies in the absence of DNA damage. In a search for interactions between Chk2 and proteins with similar subcellular localization patterns, we found that Chk2 coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome segregation, mitotic entry, and mitotic exit. Plk1 overexpression enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates recombinant Chk2 in vitro. Indirect immunofluorescence (IF) microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes in early mitosis and to the midbody in late mitosis. These findings suggest lateral communication between the DNA damage and mitotic checkpoints.
The extracellular matrix (ECM) is a dynamic and intricate microenvironment with excellent biophysical, biomechanical, and biochemical properties, which can directly or indirectly regulate cell proliferation, adhesion, migration, and differentiation, as well as plays key roles in homeostasis and regeneration of tissues and organs. The ECM has attracted a great deal of attention with the rapid development of tissue engineering in the field of regenerative medicine. Tissue-derived ECM scaffolds (also referred to as decellularized tissues and whole organs) are considered a promising therapy for the repair of musculoskeletal defects, including those that are widely used in orthopedics, although there are a few shortcomings. Similar to tissue-derived ECM scaffolds, cell-derived ECM scaffolds also have highly advantageous biophysical and biochemical properties, in particular their ability to be produced in vitro from a number of different cell types. Furthermore, cell-derived ECM scaffolds more closely resemble native ECM microenvironments. The products of cell-derived ECM have a wide range of biomedical applications; these include reagents for cell culture substrates and biomaterials for scaffolds, hybrid scaffolds, and living cell sheet coculture systems. Although cell-derived ECM has only just begun to be investigated, it has great potential as a novel approach for cell-based tissue repair in orthopedic tissue engineering. This review summarizes and analyzes the various types of cell-derived ECM products applied in cartilage, bone, and nerve tissue engineering in vitro or in vivo and discusses future directions for investigation of cell-derived ECM.
Segmented filamentous bacteria (SFB) are commensal organisms that grow by anchoring a specialized holdfast structure to the intestinal walls of a variety of animals. Interaction of SFB with Peyer’s patches in mice promotes the post-natal maturation of the immune system. We previously reported that the colonization of SFB in humans mainly occurs by 36 months of age, and is difficult to be detected afterward. In this study, we measured the level of SFB in intestinal fluids of human children. SFB were found via qPCR to represent a small fraction of the whole SFB-positive microbiota (105 SFB in 1011 total bacteria). Bacteria with filamentous segmented morphology were observed in intestinal fluids via fluorescent in situ hybridization, and from gut biopsies via scanning electron microscopy. SFB-specific DNA and peptide fragments were also identified via multiple displacement amplification PCR and mass spectrometry. There was an overall positive correlation between the presence of SFB and the titer of total secretory immunoglobulin A (sIgA), which is more apparent in intestinal fluids of the age group of 8–36 months. Afterward there was a decline of SFB in numbers correlated with a reduction of total sIgA. RT-qPCR analysis of the terminal ileal biopsies revealed that the expression of Th17 pathway genes were induced in SFB-positive samples, while the markers of T and B cell receptor signaling pathways were also upregulated. Collectively, these data suggest that SFB is a rare member of microbiota, and may play an important role in the development of human gut immunity.
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